Method Description

Fresh tissue samples (approximately 2 x 2 x 5 mm) are placed in a polymerase chain reaction (PCR) Bead Tube containing Tris-EDTA buffer, and proteinase K and digested on the Bertin Precellys Disruptor. Formalin-fixed, paraffin-embedded (FFPE) tissue scrolls are placed in a PCR bead tube containing Tris-EDTA buffer and proteinase K and undergo a prolonged heat treatment for digestion. Once digested, samples undergo DNA extraction on a MagNA Pure 96 instrument. The PCR assay employs a target-specific detection system including primers as well TaqMan detection probes alongside a SimpleProbe for melt curve analysis-based genotyping targeting the 23S ribosomal RNA gene.

 

The LightCycler 480 II instrument is used to amplify and monitor target nucleic acid sequences by fluorescence during PCR cycling. Detection of amplified product is based on the TaqMan probe principle. For PCR product detection, the TaqMan probe binds the complementary strand of amplified target. Specific PCR Taq polymerase with 5'-3' exonuclease activity degrades the probe, releasing the fluorophore and breaking the proximity to the quencher molecule, allowing fluorescence of the fluorophores. At the conclusion of PCR cycling, amplified product is thermally denatured and then cooled to allow for a fluorescein labeled SimpleProbe to anneal to an 18-base pair region of the amplified target that includes 2 positions’ mutations which are known to confer clarithromycin resistance. The temperature is slowly raised, while consistently monitoring fluorescence. The process is completed in a closed system to mitigate contamination. Contamination control is enhanced using uracil -DNA glycosylase enzymatic treatment and a master mix which includes dUTPs.(Chen D, Cunningham SA, Cole NC, Kohner PC, Mandrekar JN, Patel R. Phenotypic and molecular antimicrobial susceptibility of Helicobacter pylori. Antimicrob Agents Chemother. 2017;61(4):e02530-16. doi: 10.1128/AAC.02530-16)